Method of improving the properties of a flour dough, a flour dough improving composition and improved food products

ABSTRACT

A method of improving the Theological and/or machineability properties of a flour dough and/or the quality of the product made from the dough, comprising adding to the dough a combination comprising a Hox and an emulsifying agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of the filingdate of U.S. Provisional Patent Application No. 60/398,020, filed Jul.24, 2002. This application is also a continuation-in-part of U.S. patentapplication Ser. No. 09/932,923, filed Aug. 21, 2001, which is acontinuation of application Ser. No. 08/676,186 filed Jul. 12, 1996, nowU.S. Pat. No. 6,358,543, which was a continuation-in-part of applicationSer. No. 08/483,870, filed Jun. 7, 1995, abandoned. The Ser. No.08/483,870 application was a U.S. national phase of PCT/DK96/00239,filed Jun. 4, 1996. This application is additionally acontinuation-in-part of application Ser. No. 10/040,394, filed Jan. 9,2002, which was a divisional of application Ser. No. 09/402,664, filedOct. 22, 1999, now U.S. Pat. No. 6,406,723. U.S. Pat. No. 6,406,723 wasthe U.S. national phase of PCT/DK98/00136, filed Apr. 3, 1998, whichclaimed priority from DK0400/97, filed Apr. 9, 1997. This application isalso a continuation-in-part of U.S. application Ser. No. 10/150,429,filed May 17, 2002, which claims priority from UK Application 0112226.6,filed May 18, 2001, and U.S. Application No. 60/347,007, filed Jan. 9,2002. We claim the benefit of priority dates of all the aboveapplications. This application also claims the benefit of priority ofthe filing date of Great Britain Application 0211975.8, filed May 24,2002. The contents of all of the above applications are incorporatedherein by reference to the extent they are consistent with thisapplication and inventions described herein.

FIELD OF INVENTION

[0002] The invention pertains to the provision of flour doughs havingimproved rheological properties and farinaceous food products havingimproved quality characteristics and it provides a maltose oxidizingoxidoreductase-containing compo-sition capable of conferring suchimproved properties on doughs and finished food products made herefromwhen it is added as a component to the doughs, and a method of preparingimproved doughs and farinaceous food products.

[0003] More in particular, the present invention relates to the field offood manufacturing, in particular to the preparation of improved bakeryproducts and other farinaceous food products. Specifically, theinvention concerns the use of a new combination for improving thestability and/or machineability of dough and/or improving the quality ofbaked and dried products made from such doughs.

[0004] In a preferred aspect, the present invention relates to:

[0005] a method of improving the rheological and/or machineabilityproperties of a flour dough and/or the quality of the product made fromthe dough, comprising adding to the dough a combination comprising a Hoxand an emulsifying agent.

[0006] Teachings relating to this preferred aspect now follow.

TECHNICAL BACKGROUND AND PRIOR ART

[0007] The invention relates in particular to a method of providingflour doughs having improved Theological properties and to finishedbaked or dried products made from such doughs, which have improvedtextural, eating quality and dimensional characteristics.

[0008] In this connection, the “strength” or “weakness” of doughs is animportant aspect of making farinaceous finished products from doughs,including baking. The “strength” or “weakness” of a dough is primarilydetermined by its content of protein and in particular the content andthe quality of the gluten protein is an important factor in thatrespect. Flours with a low protein content are generally characterizedas “weak”. Thus, the cohesive, extensible, rubbery mass which is formedby mixing water and weak flour will usually be highly extensible whensubjected to stress, but it will not return to its original dimensionswhen the stress is removed.

[0009] Flours with a high protein content are generally characterized as“strong” flours and the mass formed by mixing such a flour and waterwill be less extensible than the mass formed from a weak flour, andstress which is applied during mixing will be restored without breakdownto a greater extent than is the case with a dough mass formed from aweak flour.

[0010] Strong flour is generally preferred in most baking contextsbecause of the superior Theological and handling properties of the doughand the superior form and texture qualities of the finished baked ordried products made from the strong flour dough.

[0011] Dough quality may be largely dependent on the type or types offlour present in the dough and/or the age of the flour or flours.

[0012] Doughs made from strong flours are generally more stable.Stability of a dough is one of the most important characteristics offlour doughs. According to American Association of Cereal Chemists(AACC) Method 36-0lA the term “stability” can be defined as “the rangeof dough time over which a positive response is obtained and thatproperty of a rounded dough by which it resists flattening under its ownweight over a course of time”. According to the same method, the term“response” is defined as “the reaction of dough to a known and specificstimulus, substance or set of conditions, usually determined by bakingit in comparison with a control”

[0013] Within the bakery and milling industries it is known to use dough“conditioners” to strengthen the dough to increase its stability andstrength. Such dough conditioners are normally non-specific oxidizingagents such as eg iodates, peroxides, ascorbic acid, K-bromate orazodi-carbonamide and they are added to dough with the aims of improvingthe baking performance of flour to achieve a dough with improvedstretchability and thus having a desirable strength and stability. Themechanism behind this effect of oxidizing agents is that the flourproteins, in particular gluten contains thiol groups which, when theybecome oxidized, form disulphide bonds whereby the protein forms a morestable matrix resulting in a better dough quality and improvements ofthe volume and crumb structure of the baked products.

[0014] In addition to the above usefulness of ascorbic acid/ascorbate asa dough conditioner due to its oxidizing capacity, these compounds mayalso act as substrate for an oxidoreductase, ascorbate oxidase which isdisclosed in EP 0 682 116 A1. In the presence of its substrate, thisenzyme converts ascorbic acid/ascorbate to dehydroascorbic acid andH₂O₂. This prior art does not suggest that ascorbic acid oxidase in thepresence of ascorbic acid/ascorbate might have a dough conditioningeffect, but assumingly this is the case.

[0015] However, the use of several of the currently available oxidizingagents is either objected to by consumers or is not permitted byregulatory bodies and accordingly, it has been attempted to findalternatives to these conventional flour and dough additives and theprior art has i.a. suggested the use of glucose oxidase for thispurpose. In addition, the prior art has inter alia (i.a.) suggested theuse of oxidoreductases such as carbohydrate oxidase, glycerol oxidaseand hexose oxidase for this purpose.

[0016] Thus, U.S. Pat. No. 2,783,150 discloses the addition of glucoseoxidase to flour to improve dough strength and texture and appearance ofbaked bread.

[0017] CA 2,012,723 discloses bread improving compositions comprisingcellulolytic enzymes such as xylanases and glucose oxidase, the latterenzyme being added to reduce certain disadvantageous effects of thecellulolytic enzymes (reduced dough strength and stickiness) and it isdisclosed that addition of glucose to the dough is required to obtain asufficient glucose oxidase activity.

[0018] JP-A-92-084848 suggests the use of a bread improving compositioncomprising glucose oxidase and lipase.

[0019] EP-Bl-321 811 discloses the use of an enzyme compositioncomprising sulfhydryl oxidase and glucose oxidase to improve therheological characteristics of doughs. It is mentioned in this prior artdocument that the use of glucose oxidase alone has not been successful.

[0020] In EP-B1-338 452 is disclosed an enzyme composition for improvingdough stability, comprising a mixture of cellulase/hemicellulase,glucose oxidase and optionally sulfhydryl oxidase.

[0021] However, the use of glucose oxidase as a dough improving additivehas the limitation that this enzyme requires the presence of sufficientamounts of glucose as substrate in order to be effective in a doughsystem and generally, the glucose content in cereal flours is low.Therefore, the absence of glucose in doughs or the low content hereof indoughs will be a limiting factor for the effectiveness of glucoseoxidase as a dough improving agent.

[0022] In contrast hereto, the content of maltose in cereal flours isgenerally significantly higher than that of glucose and accordingly, afreshly prepared dough will normally contain more maltose than glucose.Thus, in an experiment where the content of sugars in supernatants fromsuspensions of wheat flour and a dough prepared from the flour andfurther comprising water, yeast, salt and sucrose (as described in thefollowing example 2.3) were analyzed, the following values (% by weightcalculated on flour) were found: Flour Dough Sucrose 0.3 <0.01 Galactose0.001 0.01 Glucose 0.25 0.72 Maltose 2.6 1.4 Fructose 0.08 0.67 Lactose<0.01 <0.01

[0023] In addition, the content of maltose remains at a relatively highlevel in a dough which is leavened by yeast, since the yeast primarilyutilizes glucose, or it may even increase in the dough e.g. duringproofing due to the activity of starch degrading enzymes such as e.g.β-amylase, which is inherently present in the flour or is added to thedough.

[0024] Whereas the prior art has recognized the useful improving effectsof glucose oxidase on the rheological characteristics of bread doughsand on the quality of the corresponding baked products, it has also beenrealized that the use of this enzyme has several drawbacks. Thus, it maybe required to add sucrose or glucose as substrate to the dough toobtain a sufficient effect and glucose oxidase does not constantlyprovide a desired dough or bread improving effect when used alonewithout the addition of other enzymes.

[0025] However, it has now been found that the addition of anoxidoreductase, which is capable of oxidizing maltose, including hexoseoxidase as a sole dough conditioning agent, i.e. without concomitantaddition of substrate for the added enzyme, or of other enzymes, to afarinaceous dough results in an increased resistance hereof to breakingwhen the dough is stretched, i.e. this enzyme confers in itself to thedough an increased strength whereby the dough becomes less prone tomechanical deformation. It is contemplated that this effect of additionof hexose oxidase to a dough is the result of the formation ofcross-links between thiol groups in sulphur-containing amino acids inwheat gluten which occurs when the H₂O₂ generated by the enzyme in thedough reacts with the thiol groups which are hereby oxidized.

[0026] Hexose oxidase (D-hexose: O₂-oxidoreductase, EC 1.1.3.5) is anenzyme which in the presence of oxygen is capable of oxidizing D-glucoseand several other reducing sugars including maltose, glucose, lactose,galactose, xylose, arabinose and cellobiose to their correspondinglactones with subsequent hydrolysis to the respective aldobionic acids.Accordingly, hexose oxidases differ from glucose oxidase which can onlyconvert D-glucose, in that hexose oxidases can utilize a broader rangeof sugar substrates. The oxidation catalyzed by the enzyme can beillustrated as follows:

[0027] D-Glucose+O₂->δ-D-gluconolactone+H₂O₂, or

[0028] D-Galactose+O₂->γ-D-galactogalactone+H₂O₂

[0029] Hexose oxidase (in the following also referred to as HOX) hasbeen isolated from several red algal species such as Iridophycusflaccidum (Bean and Hassid, 1956, J. Biol. Chem., 218:425-436) andChondrus crispus (Ikawa 1982, Methods Enzymol., 89:145-149).Additionally, the algal species Euthora cristata (Sullivan et al. 1973,Biochemica et Biophysica Acta, 309:11-22) has been shown to produce HOX.

[0030] Other potential sources of hexose oxidase according to theinvention include microbial species or land-growing plant species. Thus,as an example of such a plant source, Bean et al., Journal of BiologicalChemistry (1961) 236: 1235-1240, have disclosed an oxidoreductase fromcitrus fruits which is capable of oxidizing a broad range of sugarsincluding D-glucose, D-galactose, cellobiose, lactose, maltose,D-2-deoxyglucose, D-mannose, D-glucosamine and D-xylose. Another exampleof an enzyme having hexose oxidase activity is the enzyme system ofMalleomyces mallei disclosed by Dowling et al., Journal of Bacteriology(1956) 72:555-560.

[0031] It has been reported that hexose oxidase isolated from the abovenatural sources may be of potential use in the manufacturing of certainfood products. Thus, hexose oxidase isolated from Iridophycus flaccidumhas been shown to be capable of converting lactose in milk with theproduction of the corresponding aldobionic acid and has been shown to beof potential interest as an acidifying agent in milk, e.g. to replaceacidifying microbial cultures for that purpose (Rand, 1972, Journal ofFood Science, 37:698-701). In that respect, hexose oxidase has beenmentioned as a more interesting enzyme than glucose oxidase, since thislatter enzyme can only be enzymatically effective in milk or other foodproducts not containing glucose or having a low content of glucose, ifglucose or the lactose-degrading enzyme lactase which convert thelactose to glucose and galactose, is also added.

[0032] The capability of oxidoreductases including that of hexoseoxidase to generate hydrogen peroxide has also been utilized to improvethe storage stability of certain food products including cheese, butterand fruit juice as it is disclosed in JP-B-73/016612. It has also beensuggested that oxidoreductases may be potentially useful as antioxidantsin food products.

[0033] However, the present invention has demonstrated that hexoseoxidase is highly useful as a dough conditioning agent in themanufacturing of flour dough products including not only bread productsbut also other products made from flour doughs such as noodles andalimentary paste products.

[0034] WO 94/04035 discloses a method of improving properties of a dough(with and without fat) and/or baked product made from dough by adding alipase of microbial origin to the dough. The use of the microbial lipaseresulted in an increased volume and improved softness of the bakedproduct. Furthermore an antistaling effect was found.

[0035] EP 1 108 360 A1 discloses a method of preparing a flour dough.The method comprises adding to the dough components an enzyme that underdough conditions is capable of hydrolysing a nonpolar lipid, aglycolipid and a phospholipid, or a composition containing said enzymeand mixing the dough components to obtain the dough.

[0036] WO 02/03805 discloses that the addition to dough of a combinationof two lipases with different substrate specificities. The combinationproduces a synergistic effect on the dough or on a baked product madefrom the dough. Optionally, an additional enzyme may be used togetherwith the lipase.

SUMMARY OF THE INVENTION

[0037] Accordingly, the invention relates in a first aspect to a methodof improving the rheological properties of a flour dough and the qualityof the finished product made from the dough, comprising adding to thedough ingredients, dough additives or the dough an effective amount ofan oxidoreductase which at least is capable of oxidizing maltose, suchas e.g. a hexose oxidase.

[0038] In a further aspect, there is also provided a dough bakeryproduct improving composition comprising an oxidoreductase which atleast is capable of oxidizing maltose, and at least one further doughingredient or dough additive.

[0039] In still further aspects, the invention pertains to a method ofpreparing a bakery product, comprising preparing a flour dough includingadding an effective amount of an oxidoreductase which at least iscapable of oxidizing maltose and baking the dough, and a method ofpreparing a dough-based food product comprising adding to the dough aneffective amount of a maltose oxidizing oxidoreductase.

[0040] In addition, we have surprisingly found that a combination of aHox and an emulsifying agent results in particularly advantageousproperties in dough and dough products and/or in baked productstherefrom. In particular the stability (e.g. shock stability) and/orrheological (e.g. decrease in stickiness) and/or machineabilityproperties and/or the resultant volume of either the dough and/or bakedproducts (e.g. baked products with better crumb structure and/orhomogeneity) is/are improved. Furthermore, the combination of the Hoxand emulsifying agent results in an improvement in bread quality, inparticular in respect of specific volume and/or crumb homogeneity, whichis not a simple additive effect, but may reflect a synergistic effect ofthese types of enzymes.

[0041] The invention further relates to the use of a Hox and anemulsifying agent to improve the rheological and/or machineabilityproperties of dough.

[0042] The invention further relates to the use of a Hox and anemulsifying agent to improve the volume of a baked product made from adough.

DETAILED DISCLOSURE OF THE INVENTION

[0043] In one aspect, the present method contemplates a method ofimproving the rheological properties of flour doughs. The methodcomprises, as it is mentioned above, the addition of an effective amountof a maltose oxidizing oxidoreductase either to a component of the doughrecipe or to the dough resulting from mixing all of the components forthe dough. In the present context, “an effective amount” is used toindicate that the amount is sufficient to confer to the dough and/or thefinished product improved characteristics as defined herein.

[0044] In another aspect the invention provides a method of improvingthe rheological and/or machineability properties of a flour dough and/orthe quality (e.g. volume) of the product made from the dough, comprisingadding to the dough a combination comprising a Hox and an emulsifyingagent.

[0045] Factors which influence the Theological properties and/or themachineability include stickiness and extensibility.

[0046] In another aspect the invention provides a method of improvingthe Theological and/or machineability properties of a flour dough and/orthe quality (e.g. volume) of the product made from the dough, comprisingadding to the dough a combination comprising a Hox and an emulsifyingagent wherein the flour dough comprises at least one further doughadditive or ingredient.

[0047] In another aspect the invention provides a method of improvingthe Theological and/or machineability properties of a flour dough and/orthe quality (e.g. volume) of the product made from the dough, comprisingadding to the dough a combination comprising a Hox and an emulsifyingagent wherein the product is selected from the group consisting of abread product, a noodle product, a cake product, a pasta product and analimentary paste product.

[0048] In another aspect the invention provides a method of improvingthe Theological and/or machineability properties of a flour dough and/orthe quality (e.g. volume) of the product made from the dough, comprisingadding to the dough a combination comprising a Hox and an emulsifyingagent wherein at least one further enzyme is added to the doughingredients, dough additives or the dough.

[0049] In another aspect the invention provides a dough improvingcomposition comprising a Hox and an emulsifying agent.

[0050] In another aspect the invention provides a dough improvingcomposition comprising a Hox and an emulsifying agent wherein the flourdough comprises at least one further dough additive or ingredient.

[0051] In another aspect the invention provides use of a dough improvingcomposition comprising a Hox and an emulsifying agent in the manufactureof a product made from dough wherein the product is selected from thegroup consisting of a bread product, a noodle product, a cake product, apasta product and an alimentary paste product.

[0052] In another aspect the invention provides a dough improvingcomposition comprising a Hox and an emulsifying agent wherein at leastone further enzyme is added to the dough ingredients, dough additives orthe dough.

[0053] In another aspect the invention provides use of a dough improvingcomposition comprising a Hox and an emulsifying agent wherein saidcomposition improves the Theological and/or machineability properties offlour dough.

[0054] In another aspect the invention provides use of a dough improvingcomposition comprising a Hox and an emulsifying agent wherein saidcomposition improves the volume of a baked product made from a flourdough.

[0055] In another aspect the invention provides a dough for addition toa sponge wherein said dough comprises a Hox and an emulsifying agent.

[0056] In another aspect the invention provides a dough for addition toa sponge wherein said dough comprises a Hox and an emulsifying agent andwherein the dough comprises at least one further dough additive oringredient.

[0057] Hexose Oxidase

[0058] In one useful embodiment of the method according to theinvention, the oxidoreductase is a hexose oxidase.

[0059] The term “Hox” as used herein refers to Hexose oxidase(D-hexose:O₂-oxidoreductase, EC 1.1.3.5). Below discloses some of thesources of Hox. WO 96/40935 discloses a method of producing Hox byrecombinant DNA technology. U.S. Pat. No. 6,251,626 discloses hexoseoxidase sequences.

[0060] The Hox may be isolated and/or purified from natural sources orit may be prepared by use of recombinant DNA techniques.

[0061] The Hox may be a variant or derivative of a natural Hox.

[0062] The Hox, or the variant or derivative of a natural Hox, iscapable of oxidising maltose in the dough.

[0063] Preferably the Hox is added in a substantially pure and/orsubstantially isolated form.

[0064] Hexose oxidase can, as it is described in details herein, beisolated from marine algal species naturally producing that enzyme. Suchspecies are found in the family Gigartinaceae which belong to the orderGigartinales. Examples of hexose oxidase producing algal speciesbelonging to Gigartinaceae are Chondrus crispus and Iridophycusflaccidum. Also algal species of the order Cryptomeniales including thespecies Euthora cristata are potential sources of hexose oxidase.

[0065] When using such natural sources for hexose oxidase, the enzyme istypically isolated from the algal starting material by extraction usingan aqueous extraction medium. As starting material may be used algae intheir fresh state as harvested from the marine area where they grow, orthe algal material can be used for extraction of hexose oxidase afterdrying the fronds e.g. by air-drying at ambient temperatures or by anyappropriate industrial drying method such as drying in circulated heatedair or by freeze-drying. In order to facilitate the subsequentextraction step, the fresh or dried starting material may advantageouslybe comminuted e.g. by grinding or blending.

[0066] As the aqueous extraction medium, buffer solutions e.g. having apH in the range of 5-8, such as 0.1 M sodium phosphate buffer, 20 mMtriethanolamine buffer or 20 mM Tris-HCl buffer are suitable. The hexoseoxidase is typically extracted from the algal material by suspending thestarting material in the buffer and keeping the suspension at atemperature in the range of 0-20° C. such as at about 5° C. for 1 to 5days, preferably under agitation.

[0067] The suspended algal material is then separated from the aqueousmedium by an appropriate separation method such as filtration, sievingor centrifugation and the hexose oxidase is subsequently recovered fromthe filtrate or supernatant. Optionally, the separated algal material issubjected to one or more further extraction steps.

[0068] Since several marine algae contain coloured pigments such asphycocyanins, it may be required to subject the filtrate or supernatantto a further purification step whereby these pigments are removed. As anexample, the pigments may be removed by treating the filtrate orsupernatant with an organic solvent in which the pigments are solubleand subsequently separating the solvent containing the dissolvedpigments from the aqueous medium. Alternatively, pigments may be removedby subjecting the filtrate or supernatant to a hydrophobic interactionchromatography step.

[0069] The recovery of hexose oxidase from the aqueous extraction mediumis carried out by any suitable conventional methods allowing isolationof proteins from aqueous media. Such methods, examples of which will bedescribed in details in the following, include conventional methods forisolation of proteins such as ion exchange chromatography, optionallyfollowed by a concentration step such as ultrafiltration. It is alsopossible to recover the enzyme by adding substances such as e.g.(NH₄)₂SO₄ or polyethylene glycol (PEG) which causes the protein toprecipitate, followed by separating the precipitate and optionallysubjecting it to conditions allowing the protein to dissolve.

[0070] For certain applications of hexose oxidase it is desirable toprovide the enzyme in a substantially pure form e.g. as a preparationessentially without other proteins or non-protein contaminants andaccordingly, the relatively crude enzyme preparation resulting from theabove extraction and isolation steps may be subjected to furtherpurification steps such as further chromatography steps, gel filtrationor chromato-focusing as it will also be described by way of example inthe following.

[0071] Further Dough Additives or Ingredients (Components)

[0072] In a preferred embodiment of the method according to theinvention, a flour dough is prepared by mixing flour with water, aleavening agent such as yeast or a conventional chemical leaveningagent, and an effective amount of hexose oxidase under dough formingconditions. It is, however, within the scope of the invention thatfurther components can be added to the dough mixture.

[0073] Typically, such further dough components include conventionallyused dough components such as salt (such as sodium chloride, calciumacetate, sodium sulfate or calcium sulfate), sweetening agents such assugars, syrups or artificial sweetening agents, lipid substancesincluding shortening, margarine, butter or an animal or vegetable oil,glycerol and one or more dough additives such as emulsifying agents,starch degrading enzymes, cellulose or hemicellulose degrading enzymes,proteases, lipases, non-specific oxidizing agents such as thosementioned above, flavouring agents, lactic acid bacterial cultures,vitamins, minerals, hydrocolloids such as alginates, carrageenans,pectins, vegetable gums including e.g. guar gum and locust bean gum, anddietary fiber substances.

[0074] The dough may also comprise other conventional dough ingredients,e.g.: proteins, such as milk powder, gluten, and soy; eggs (either wholeeggs, egg yolks or egg white); an oxidant such as ascorbic acid,potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammoniumpersulfate; an amino acid such as L-cysteine; a sugar.

[0075] The dough may comprise fat such as granulated fat or shortening.

[0076] The further dough additive or ingredient can be added togetherwith any dough ingredient including the flour, water or optional otheringredients or additives, or the dough improving composition. Thefurther dough additive or ingredient can be added before the flour,water, optional other ingredients and additives or the dough improvingcomposition. The further dough additive or ingredient can be added afterthe flour, water, optional other ingredients and additives or the doughimproving composition.

[0077] The further dough additive or ingredient may conveniently be aliquid preparation. However, the further dough additive or ingredientmay be conveniently in the form of a dry composition.

[0078] Preferably the further dough additive or ingredient is selectedfrom the group consisting of a vegetable oil, a vegetable fat, an animalfat, shortening, glycerol, margarine, butter, butterfat and milk fat.

[0079] Preferably the further dough additive or ingredient is at least1% the weight of the flour component of dough. More preferably, thefurther dough additive or ingredient is at least 2%, preferably at least3%, preferably at least 4%, preferably at least 5%, preferably at least6%.

[0080] If the additive is a fat, then typically the fat may be presentin an amount of from 1 to 5%, typically 1 to 3%, more typically about2%.

[0081] Further Enzymes

[0082] In one advantageous embodiment of the above method at least onefurther enzyme is added to the dough. Suitable examples hereof include acellulase, a hemicellulase, a xylanase, a starch degrading enzyme, aglucose oxidase, a lipase, a lipoxygenase, an oxidoreductase and aprotease.

[0083] Among starch degrading enzymes, amylases are particularly usefulas dough improving additives. Other useful starch degrading enzymeswhich may be added to a dough composition include glucoamylases andpullulanases.

[0084] The term “xylanase” as used herein refers to xylanases (EC3.2.1.32) which hydrolyse xylosidic linkages.

[0085] The further enzyme can be added together with any doughingredient including the flour, water or optional other ingredients oradditives, or the dough improving composition. The further enzyme can beadded before the flour, water, and optionally other ingredients andadditives or the dough improving composition. The further enzyme can beadded after the flour, water, and optionally other ingredients andadditives or the dough improving composition.

[0086] The further enzyme may conveniently be a liquid preparation.However, the composition may be conveniently in the form of a drycomposition.

[0087] In some aspects of the present invention it may be found thatsome enzymes of the dough improving composition of the invention arecapable of interacting with each other under the dough conditions to anextent where the effect on improvement of the rheological and/ormachineability properties of a flour dough and/or the quality of theproduct made from dough by the enzymes is not only additive, but theeffect is synergistic.

[0088] In relation to improvement of the product made from dough(finished product), it may be found that the combination results in asubstantial synergistic effect in respect to crumb homogeneity asdefined herein. Also, with respect to the specific volume of bakedproduct a synergistic effect may be found.

[0089] Emulsifying Agent

[0090] The dough may further comprise a further emulsifier such as mono-or diglycerides, sugar esters of fatty acids, polyglycerol esters offatty acids, lactic acid esters of monoglycerides, acetic acid esters ofmonoglycerides, polyoxethylene stearates, or lysolecithin. Among starchdegrading enzymes, amylases are particularly useful as dough improvingadditives. α-amylase breaks down starch into dextrins which are furtherbroken down by β-amylase into maltose. Other useful starch degradingenzymes which may be added to a dough composition include glucoamylasesand pullulanases. In the present context, further interesting enzymesare xylanases and other oxidoreductases such as glucose oxidase,pyranose oxidase and sulfhydryl oxidase.

[0091] Conventional emulsifiers used in making flour dough productsinclude as examples monoglycerides, diacetyl tartaric acid esters ofmono- and diglycerides of fatty acids, and lecithins e.g. obtained fromsoya.

[0092] The emulsifying agent may be an emulsifier per se or an agentthat generates an emulsifier in situ.

[0093] Examples of emulsifying agents that can generate an emulsifier insitu include enzymes.

[0094] Preferably the emulsifying agent is a lipase.

[0095] Lipase

[0096] The term “lipase” as used herein refers to enzymes which arecapable of hydrolysing carboxylic ester bonds to release carboxylate (EC3.1.1). Examples of lipases include but are not limited totriacylglycerol lipase (EC 3.1.1.3), galactolipase (EC 3.1.1.26),phospholipase (EC 3.1.1.32).

[0097] The lipase may be isolated and/or purified from natural sourcesor it may be prepared by use of recombinant DNA techniques.

[0098] Preferably the lipase is selected from the group comprisingtriacylglycerol lipase, a galactolipase, phospholipase.

[0099] More preferably the lipase(s) may be one or more of:triacylglycerol lipase (EC 3.1.1.3), phospholipase A2 (EC 3.1.1.4),galactolipase (EC 3.1.1.26), phospholipase A1 (EC 3.1.1.32), lipoproteinlipase A2 (EC 3.1.1.34).

[0100] The lipase may be a variant or derivative of a natural lipase.

[0101] For some aspects, preferably the lipase is a phospholipase(including a variant phospholipase).

[0102] Preferably the lipase is added in a substantially pure and/orsubstantially isolated form.

[0103] Lipases that are useful in the present invention can be derivedfrom a bacterial species, a fungal species, a yeast species, an animalcell and a plant cell. Whereas the enzyme may be provided by cultivatingcultures of such source organisms naturally producing lipase, it may bemore convenient and cost-effective to produce it by means of geneticallymodified cells such as it is described WO 9800136. The term “derived”may imply that a gene coding for the lipase is isolated from a sourceorganism and inserted into a host cell capable of expressing the gene.

[0104] WO 02/03805 teaches some of the sources of lipases. The lipasesthat are taught therein are incorporated herein by reference.

[0105] For some aspects of the present invention the lipase may beLipopan F (supplied by Novozymes) or a variant thereof.

[0106] Dough Preparation

[0107] The dough is prepared by admixing flour, water, theoxidoreductase according to the invention or the dough improvingcomposition and optionally other possible ingredients and additives. Theoxidoreductase or dough improving composition can be added together withany dough ingredient including the flour, the water or dough ingredientmixture or with any additive or additive mixture. The dough improvingcomposition can be added before the flour or water or optional otheringredients and additives. The dough improving composition can be addedafter the flour or water, or optional other ingredients and additives.The dough can be prepared by any conventional dough preparation methodcommon in the baking industry or in any other industry making flourdough based products.

[0108] The dough of the invention generally comprises wheat meal orwheat flour and/or other types of meal, flour or starch such as cornflour, corn starch, maize flour, rice flour, rye meal, rye flour, oatflour, oat meal, soy flour, sorghum meal, sorghum flour, potato meal,potato flour or potato starch.

[0109] A preferred flour is wheat flour, but doughs comprising flourderived from other cereal species such as from rice, maize, corn, oat,barley, rye, durra, soy, sorghum and potato are also contemplated.

[0110] Preferably the flour dough comprises a hard flour.

[0111] The term “hard flour” as used herein refers to flour which has ahigher protein content such as gluten than other flours and is suitablefor the production of, for example, bread. The term “hard flour” as usedherein is synonymous with the term “strong flour”.

[0112] Preferably the flour dough comprises a hard wheat flour.

[0113] The invention also provides a pre-mix comprising flour togetherwith the combination as described herein. The pre-mix may contain otherdough-improving and/or bread-improving additives, e.g. any of theadditives, including enzymes, mentioned herein.

[0114] The dough of the invention may be fresh, frozen, or part-baked.

[0115] The dough of the invention can be a leavened dough or a dough tobe subjected to leavening. The dough may be leavened in various ways,such as by adding chemical leavening agents, e.g., sodium bicarbonate orby adding a leaven (fermenting dough), but it is preferred to leaven thedough by adding a suitable yeast culture, such as a culture ofSaccharomyces cerevisiae (baker's yeast), e.g. a commercially availablestrain of S. cerevisiae.

[0116] The oxidoreductase or the dough improving composition can beadded as a liquid preparation or in the form of a dry powder compositioneither comprising the enzyme as the sole active component or inadmixture with one or more other dough ingredients or additive.

[0117] The amount of the enzyme component added normally is an amountwhich results in the presence in the finished dough of 1 to 10,000 unitsper kg of flour, preferably 5 to 5000 units such as 10 to 1000 units. Inuseful embodiments, the amount is in the range of 20 to 500 units per kgof flour. In the present context 1 oxidoreductase unit corresponds tothe amount of enzyme which under specified conditions results in theconversion of 1 μmole glucose per minute. The activity is stated asunits per g of enzyme preparation.

[0118] Rheological Properties

[0119] The phrase “Theological properties” as used herein relates to thephysical and chemical phenomena described herein which in combinationwill determine the performance of flour doughs and thereby also thequality of the resulting products.

[0120] The phrase “machineability of a flour dough” as used hereinrefers to the improved manipulation by machinery of the dough. The doughis less sticky compared to the dough without the addition of thecombination.

[0121] In a further embodiment, the invention relates to improvement ofthe Theological characteristics of the dough including that the glutenindex in the dough is increased by at least 5%, relative to a doughwithout addition of a combination, the gluten index is determined bymeans of a Glutomatic 2200 apparatus.

[0122] The phrase “rheological properties” as used herein refers to theeffects of dough conditioners on dough strength and stability as themost important characteristics of flour doughs. According to AmericanAssociation of Cereal Chemists (AACC) Method 36-01A the term “stability”can be defined as “the range of dough time over which a positiveresponse is obtained and that property of a rounded dough by which itresists flattening under its own weight over a course of time”.According to the same method, the term “response” is defined as “thereaction of dough to a known and specific stimulus, substance or set ofconditions, usually determined by baking it in comparison with acontrol”.

[0123] As it is mentioned herein, it is generally desirable to improvethe baking performance of flour to achieve a dough with improvedstretchability and thus having a desirable strength and stability byadding oxidising agents which cause the formation of protein disulphidebonds whereby the protein forms a more stable matrix resulting in abetter dough quality and improvements of the volume and crumb structureof baked products.

[0124] The effect of the oxidoreductase or the dough improvingcomposition on the rheological properties of the dough can be measuredby standard methods according to the International Association of CerealChemistry (ICC) and the American Association of Cereal Chemistry (AACC)including the amylograph method (ICC 126), the farinograph method (AACC54-21) and the extensigraph method (AACC 54-10). The extensigraph methodmeasures e.g. the doughs ability to retain gas evolved by yeast and theability to withstand proofing. In effect, the extensigraph methodmeasures the relative strength of a dough. A strong dough exhibits ahigher and, in some cases, a longer extensigraph curve than does a weakdough. AACC method 54-10 defines the extensigraph in the followingmanner: “the extensigraph records a load-extension curve for a testpiece of dough until it breaks. Characteristics of load-extension curvesor extensigrams are used to assess general quality of flour and itsresponses to improving agents”.

[0125] In a preferred embodiment of the invention, the resistance toextension of the dough in terms of the ratio between the resistance toextension (height of curve, B) and the extensibility (length of curve,C), i.e. the B/C ratio as measured by the AACC method 54-10 is increasedby at least 10% relative to that of an otherwise similar dough notcontaining oxidoreductase. In more preferred embodiments, the resistanceto extension is increased by at least 20%, such as at least 50% and inparticular by at least 100%.

[0126] The method according to the invention can be used for any type offlour dough with the aims of improving the rheologi-cal propertieshereof and the quality of the finished prod-ucts made from theparticular type of dough. Thus, the method is highly suitable for themaking of conventional types of yeast leavened bread products includingwheat flour based bread products such as loaves and rolls. However, itis contemplated that the method also can improve the properties ofdoughs in which leavening is caused by the addition of chemicalleavening agents, including sweet bakery products such as cake productsincluding as examples pound cakes and muffins, or scones.

[0127] Noodles

[0128] In one interesting aspect, the invention is used to improve theTheological properties of doughs intended for noodle products including“white noodles” and “chinese noodles” and to improve the texturalqualities of the finished noodle products. A typical basic recipe forthe manufacturing of noodles comprises the following ingredients: wheatflour 100 parts, salt 0.5 parts and water 33 parts. Furthermore,glycerol is often added to the noodle dough. The noodles are typicallyprepared by mixing the ingredients in an appropriate mixing apparatusfollowed by rolling out the noodle dough using an appropriate noodlemachine to form the noodle strings which are subsequently air dried.

[0129] The quality of the finished noodles is assessed inter alia (i.a.)by their colour, cooking quality and texture. The noodles should cook asquickly as possible, remain firm after cooking and should preferably notloose any solids to the cooking water. On serving the noodles shouldpreferably have a smooth and firm surface not showing stickiness andprovide a firm “bite” and a good mouthfeel. Furthermore, it is importantthat the noodles have a light colour.

[0130] Since the appropriateness of wheat flour for providing noodleshaving the desired textural and eating qualities may vary according tothe year and the growth area, it is usual to add noodle improvers to thedough in order to compensate for sub-optimal quality of the flour.Typically, such improvers will comprise dietary fiber substances,vegetable proteins, emulsifiers and hydrocolloids such as e.g.alginates, carrageenans, pectins, vegetable gums including guar gum andlocust bean gum, and amylases, and glycerol.

[0131] It has been attempted to use glucose oxidase as a noodleimproving agent. However, as mentioned above, the content of glucose maybe so low in wheat flour that this enzyme will not be effective.

[0132] It is therefore an important aspect of the invention that theoxidoreductase according to the invention and the composition accordingto the invention is useful as a noodle improving agent, optionally incombination with glycerol and other components currently used to improvethe quality of noodles. Thus, it is contemplated that noodles preparedin accordance with the above method will have improved properties withrespect to colour, cooking and eating qualities including a firm,elastic and non-sticky texture and consistency.

[0133] Alimentary Paste Product

[0134] In a further useful embodiment the dough which is prepared by themethod according to the invention is a dough for preparing an alimentarypaste product. Such products which include as examples spaghetti andmaccaroni are typically prepared from a dough comprising as the mainingredients such as flour, eggs or egg powder and/or water. After mixingof the ingredient, the dough is formed to the desired type of pasteproduct and air dried. It is contemplated that the addition of thecombination to a paste dough, optionally in combination with itssubstrate, will have a significant improving effect on the extensibilityand stability hereof resulting in finished paste product having texturaland eating qualities.

[0135] In a further aspect of the invention there is provided a doughimproving composition comprising the oxidoreductase according to theinvention and at least one further dough ingredient or dough additive.

[0136] Bread

[0137] In the invention the improvement of the rheological properties ofthe dough include that the resistance to extension of the dough in termsof the ratio between resistance to extension (height of curve, B) andthe extensibility (length of curve, C), i.e. the B/C ratio, as measuredby the AACC method 54-10 is increased by at least 10% relative to thatof an otherwise similar dough that does not comprise the combination andwherein the improvement of the quality of the finished product made fromthe dough is that the average pore diameter of the crumb of the breadmade from the dough is reduced by at least 10%, relative to a breadwhich is made from a bread dough without addition of the combination.

[0138] In a further embodiment, the invention, implies that theimprovement of the quality of the product made from the dough consistsin that the pore homogeneity of the crumb of the bread made from thedough is increased by at least 5%, relative to a bread which is madefrom a bread dough without addition of the combination. The porehomogeneity of bread is conveniently measured by means of an imageanalyser composed of a standard CCD-video camera, a video digitiser anda personal computer with WinGrain software. Using such an analyzer, theresults of pore diameter in mm and pore homogeneity can be calculated asan average of measurements from 10 slices of bread. The pore homogeneityis expressed in % of pores that are larger than 0.5 times the average ofpore diameter and smaller than 2 times the average diameter.

[0139] Preferably, the dough is a yeast leavened dough. Although, it ispreferred to use the method of the present invention for the manufactureof yeast leavened bread products such as bread loaves, rolls or toastbread, the use of the method for any other type of dough and dough basedproducts such as noodle and pasta products and cakes, the quality ofwhich can be improved by the addition of the combination according tothe present invention, is also contemplated.

[0140] Preferably the method comprises a further step that the dough isbaked to obtain a baked product.

[0141] Preferably, when the dough is a bread dough, the method comprisesas a further step that the dough is baked to obtain a baked product. Oneparticularly desired property of baked bread products is a high specificvolume as defined in the examples. Accordingly, the addition of thecombination of the invention preferably results in an increase of thespecific volume of the baked product that is at least 10%, relative to abaked product made under identical conditions except that the enzyme isnot added. More preferably, the increase of the specific volume is atleast 20% such as at least 30%, e.g. at least 40%. Alternatively, thedough is a dough selected from the group consisting of a pasta dough, anoodle dough, and a cake dough or batter.

[0142] The phrase “quality of the product” as used herein refers to thefinal and stable volume and/or crust colour and/or texture and taste.

[0143] The term “product made from dough” as used herein refers to abread product such as in the form of loaves or rolls, french baguettetype bread, pita bread, tacos and crisp bread. Preferably the termrefers to cakes, pan-cakes, biscuits. More preferably the term refers topasta. More preferably the term refers to noodles. More preferably theterm refers to alimentary paste product.

[0144] In a preferred embodiment, the oxidoreductase is hexose oxidase.The further ingredients or additive can be any of the ingredients oradditives which are described above. The composition may conveniently bea liquid preparation comprising the oxidoreductase. However, thecomposition is conveniently in the form of dry composition. It will beunderstood that the amount of oxidoreductase activity in the compositionwill depend on the types and amounts of the further ingredients oradditives. However, the amount of oxidoreductase activity is preferablyin the range of 10 to 100,000 units, preferably in the range of 100 to50,000 units such as 1,000 to 10,000 units including 2,000 to 5,000units.

[0145] Optionally, the composition may be in the form of a completedough additive mixture or pre-mixture for a making a particular finishedproduct and containing all of the dry ingredients and additives for sucha dough. In specific embodiments, the composition may be oneparticularly useful for preparing a baking product or in the making of anoodle product or an alimentary paste product.

[0146] As mentioned above, the present invention provides a method forpreparing a bakery product including the addition to the dough of anoxidoreductase such as e.g. hexose oxidase. In particular, this methodresults in bakery products such as the above mentioned products in whichthe specific volume is increased relative to an otherwise similar bakeryproduct, prepared from a dough not containing oxidoreductase. It hasbeen found that the addition of the composition of the present inventionto bakery product doughs results in bakery products such as yeastleavened and chemically leavened products in which the specific volumeis increased relative to an otherwise similar bakery product. In thiscontext, the expression “specific volume” is used to indicate the ratiobetween volume and weight of the product. It has been found that, inaccordance with the method described herein, the specific volume can beincreased significantly such as by at least 10%, preferably by at least20%, including by at least 30%, preferably by at least 40% and morepreferably by at least 50%.

[0147] The present invention is highly suitable for improving theTheological and/or machineability properties and/or quality (e.g.volume) of the finished products (products made from the dough) ofconventional types of yeast leavened bread products based on wheatflour, such as loaves and rolls. The present invention is also suitablefor improving the Theological properties of doughs containing chemicalleavening agents (baking powder) and the quality (e.g. volume) ofproducts made from such doughs. Such product include as examples breads,sponge cakes and muffins.

[0148] Enzyme Amount

[0149] Preferably the or each enzyme is added in an amount from 1-1000ppm, preferably 25-500 ppm, more preferably 50-300 ppm.

[0150] Nucleotide Sequence

[0151] The enzyme need not be a native enzyme. In this regard, the term“native enzyme” means an entire enzyme that is in its native environmentand when it has been expressed by its native nucleotide sequence.

[0152] The nucleotide sequence of the present invention may be preparedusing recombinant DNA techniques (i.e. recombinant DNA). However, in analternative embodiment of the invention, the nucleotide sequence couldbe synthesised, in whole or in part, using chemical methods well knownin the art (see Caruthers M H et al (1980) Nuc Acids Res Symp Ser 215-23and Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).

[0153] Amino Acid Sequences

[0154] The enzyme may be prepared/isolated from a suitable source, or itmay be made synthetically or it may be prepared by use of recombinantDNA techniques.

[0155] Variants/Homologues/Derivatives

[0156] The present invention also encompasses the use of variants,homologues and derivatives of any amino acid sequence of an enzyme ofthe present invention or of any nucleotide sequence encoding such anenzyme. Here, the term “homologue” means an entity having a certainhomology with the subject amino acid sequences and the subjectnucleotide sequences. Here, the term “homology” can be equated with“identity”.

[0157] In the present context, an homologous sequence is taken toinclude an amino acid sequence which may be at least 75, 85 or 90%identical, preferably at least 95 or 98% identical to the subjectsequence. Typically, the homologues will comprise the same active sitesetc. as the subject amino acid sequence. Although homology can also beconsidered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of the presentinvention it is preferred to express homology in terms of sequenceidentity.

[0158] Homology comparisons can be conducted by eye, or more usually,with the aid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

[0159] The invention will now be described by way of illustration in thefollowing non-limiting examples and the drawings, in which:

[0160]FIG. 1 which is a photographic image of a bread;

[0161]FIG. 2 which is a photographic image of a bread; and

[0162]FIG. 3 which is a photographic image of a bread.

EXAMPLE 1

[0163] 1.1. Purification of Hexose Oxidase from Chondrus crispus

[0164] A purified hexose oxidase preparation was obtained using thebelow extraction and purification procedures. During these proceduresand the following characterizations of the purified enzyme, thefollowing assay for determination of hexose oxidase activity was used:

[0165] 1.1.1. Assay of Hexose Oxidase Activity

[0166] The assay was based on the method described by Sullivan and Ikawa(Biochimica et Biophysica Acta, 1973, 309:11-22), but modified to run inmicrotiter plates. An assay mixture contained 150 μl β-D-glucose (0.1 Min 0.1 M sodium phosphate buffer, pH 6.3), 120 μl 0.1 M sodium phosphatebuffer, pH 6.3, 10 μl o-dianisidine-dihydrochloride (Sigma D-3252, 3.0mg/ml in H₂O), 10 μl peroxidase (POD) (Sigma P-8125, 0.1 ml in 0.1 Msodium phosphate buffer, pH 6.3) and 10 μl enzyme (HOX) solution. Blankswere made by adding buffer in place of enzyme solution.

[0167] The incubation was started by the addition of glucose. After 15minutes of incubation at 25° C. the absorbance at 405 nm was read in anELISA reader. A standard curve was constructed using varyingconcentrations of H₂O₂ in place of the enzyme solution.

[0168] The reaction can be described in the following manner:

[0169] HOX

[0170] β-D-glucose+H₂O+O2->gluconic acid+H₂O₂

[0171] H₂O₂+β-dianisidine_(red)->2H₂O+o-dianisidine_(ox)

[0172] Oxidized o-dianisidine has a yellow colour absorbing at 405 nm.

[0173] 1.1.2. Extraction

[0174] Fresh Chondrus crispus fronds were harvested along the coast ofBrittany, France. This fresh material was homogenized in a pin mill(Alpine). To a 100 g sample of the resulting homogenized frond materialwas added 300 ml of 0.1 M sodium phosphate buffer, pH 6.8. The mixturewas subsequently sonicated in a sonication bath for 5 minutes and thenextracted under constant rotation for 4 days at 5° C., followed bycentrifugation of the mixture at 47,000×g for 20 minutes.

[0175] 300 ml of the resulting clear pink supernatant was desalted byultrafiltration using an Amicon ultrafiltration unit equipped with anOmega (10 kD cut off, Filtron) ultrafiltration membrane.

[0176] 1.1.3. Anion Exchange Step

[0177] The retentate resulting from 1.1.2 was applied to a 5×10 cmcolumn with 200 ml Q-Sepharose FF equilibrated in 20 mM triethanolamine,pH 7.3. The column was washed with the equilibration buffer and hexoseoxidase eluted with a 450 ml gradient of 0 to 1 M of NaCl inequilibration buffer. The column was eluted at 6 ml/minute, andfractions of 14 ml collected. Fractions 9-17 (total 125 ml) were pooledand concentrated by ultrafiltration using an Amicon 8400 unit equippedwith an Omega (10 kD cut off, Filtron) ultrafiltration membrane to 7.5ml.

[0178] 1.1.4. Gel Filtration

[0179] The above 7.5 ml retentate was applied to a Superdex 200 2.6×60cm gel filtration column equilibrated in 50 mM sodium phosphate buffer,pH 6.4 and eluted at a flow rate of 1 ml/-minute. Fractions of 4 ml werecollected. Fractions 17-28 (total volume 50 ml) containing the hexoseoxidase activity were pooled.

[0180] 1.1.5. Hydrophobic Interaction Chromatography

[0181] To the pool resulting from the gel filtration step 1.1.4 ammoniumsulphate was added to a final concentration of 2 M. This mixture wasthen applied to a 1.6×16 cm column with 32 ml phenyl sepharoseequilibrated in 20 mM sodium phosphate buffer, pH 6.3 and 2 M (NH₄)₂SO₄.The column was washed with equilibration buffer followed by elution ofhexose oxidase at a flow rate of 2 ml/minute using a 140 linear gradientfrom 2 M to 0 M (NH₄)₂SO₄ in 20 mM sodium phosphate buffer. Fractions of4 ml were collected and fractions 24-33 containing the hexose oxidaseactivity were pooled.

[0182] The above mentioned pink colour accompanies the enzyme, but it isseparated from hexose oxidase in this purification step.

[0183] 1.1.6. Mono Q Anion Exchange

[0184] The above pool resulting from the above phenyl sepharosechromatography step was desalted by ultrafiltration as described above.2 ml of this pool was applied to a Mono Q HR 5/5 column equilibrated in20 mM triethanolamine, pH 7.3. The column was subsequently eluted usinga 45 ml linear gradient from 0 to 0.65 M NaCl in equilibration buffer ata flow rate 1.5 ml/minute. Fractions of 1.5 ml were collected andfractions 14-24 were pooled.

[0185] 1.1.7. Mono P Anion Exchange

[0186] The hexose oxidase-containing pool from the above step 1.1.6 wasapplied to a Mono P HR 5/5 column equilibrated in 20 mM bis-Tris buffer,pH 6.5. The enzyme was eluted using a 45 ml linear gradient from 0 to0.65 M NaCl in equilibration buffer at a flow rate of 1.5 ml/minute, andfractions of 0.75 ml were collected. The highest hexose oxidase activitywas found in fraction 12.

[0187] 1.2. Characterization of the Purified Hexose Oxidase

[0188] The hexose oxidase-containing pools from the above steps 1.1.6and 1.1.7 were used in the below characterization experiments:

[0189] 1.2.1. Determination of Molecular Weight

[0190] The size of the purified native hexose oxidase was determined bygel permeation chromatography using a Superose 6 HR 10/30 column at aflow rate of 0.5 ml/minute in 50 mM sodium phosphate buffer, pH 6.4.Ferritin (440 kD), catalase (232 kD), aldolase (158 kD), bovine serumalbumin (67 kD) and chymotrypsinogen (25 kD) were used as sizestandards. The molecular weight of the purified hexose oxidase wasdetermined to be 120+10 kD.

[0191] 1.2.2. Determination of pH Optimum

[0192] Assay mixtures for the determination of pH optimum (final volume300 μl) contained 120 μl of 0.1 M stock solution of sodiumphosphate/citrate buffer of varying pH values. All other assay mixturecomponents were dissolved in H₂O. The pH was determined in the dilutedstock buffer solutions at 25° C. The hexose oxidase showed enzymaticactivity from pH 3 to pH 8, but with optimum in the range of 3.5 to 5.5.

[0193] 1.2.3. K_(m) of the Hexose Oxidase for Glucose and MaltoseRespectively

[0194] Kinetic data were fitted to V=V_(max)S/(Km+S), where V_(max) isthe maximum velocity, S is the substrate concentration and Km is theconcentration giving 50% of the maximum rate (Michaelis constant) usingthe EZ-FIT curve fitting microcomputer programme (Perrella, F. W., 1988,Analytical Biochemistry, 174:437-447).

[0195] A typical hyperbolic saturation curve was obtained for the enzymeactivity as a function of glucose and maltose, respectively. K_(m) forglucose was calculated to be 2.7 mM±0.7 mM and for maltose the K_(m) wasfound to be 43.7±5.6 mM.

EXAMPLE 2

[0196] Dough Improving Effect of Hexose Oxidase Extracted from Chondruscrispus

[0197] 2.1. Purification of Hexose Oxidase from Chondrus crispus

[0198] For this experiment, hexose oxidase was prepared in the followingmanner:

[0199] Fresh Chondrus crispus material was collected at the coast ofBrittany, France. The material was freeze-dried and subsequently ground.40 g of this ground material was suspended in 1000 ml of 20 mMtriethanolamine (TEA) buffer, pH 7.3 and left to stand at 5° C. forabout 64 hours with gentle agitation and then centrifuged at 2000×g for10 minutes. The supernatant was filtered through GF/A and GF/C glassfilters followed by filtering through a 45 μm pore size filter to obtaina filtrate preparation of 800 ml having hexose oxidase activitycorresponding to a glucose oxidase activity of 0.44 units per g ofpreparation. The activity was determined using the below procedure.

[0200] The supernatant was applied onto a 330 ml bed volumechromatographic column with anionic exchange Q Sepharose Big Beads (deadvolume 120 ml). The bound proteins were eluted over 180 minutes using agradient from 0 to 0.5 M NaCl in 20 mM TEA buffer, pH 7.3 followed by 1M NaCl in 20 mM TEA buffer, and fractions of 9 ml were collected andanalyzed for hexose oxidase activity using the below analyticalprocedure.

[0201] Hexose oxidase activity-containing fractions 60-83 were pooled(about 250 ml) and concentrated and desalted by ultrafiltration to about25 ml. This step was repeated twice on the retentates to which was added100 ml 0.05 mM TEA. The resulting retentate of 25 ml contained 0.95glucose oxidase activity units per g.

[0202] 2.2. Determination of Glucose Oxidase Activity

[0203] Definition: 1 glucose oxidase (GOD) unit corresponds to theamount of enzyme which under the specified conditions results in theconversion of 1 μmole glucose per min. The activity is stated as unitsper g of enzyme preparation.

[0204] Reagents: (i) Buffer: 20 g Na₂HPO₄-2H₂O is dissolved in 900 mldistilled water, pH is adjusted to 6.5; (ii) dye reagent (stocksolution): 200 mg of 2,6-dichloro-phenol-indophenol, Sigma No. D-1878 isdissolved in 1000 ml distilled water under vigorous agitation for 1hour; (iii) peroxidase (stock solution): Boehringer Mannheim No. 127361, 10,000 units is dissolved in 10 ml distilled water and 4.2 g ofammonium sulphate added; (iv) substrate: 10% w/v D-glucose solution inbuffer, (v) standard enzyme: hydrase #1423 from Amano.

[0205] Analytical principle and procedure: Glucose is converted togluconic acid and H₂O₂ which is subsequently converted by peroxidase toH₂O and O₂. The generated oxygen oxidizes the blue dye reagent2,6-dichloro-phenol-indophenol which thereby changes its colour topurple. The oxidized colour is measured spectrophotometrically at 590 nmand the enzymatic activity values calculated relative to a standard.

[0206] 2.3. The Effect of the Hexose Oxidase Preparation on CrosslinkingBetween Thiol Groups in a Wheat Flour Based Dough

[0207] The effect of hexose oxidase on the formation of thiol groupcross-linking was studied by measuring the content of free thiol groupsin a dough prepared from 1500 g of wheat flour, 400 Brabender Units (BU)of water, 90 g of yeast, 20 g of sucrose and 20 g of salt to which wasadded 0, 100, 250, 875 and 1250 units per kg of flour, respectively ofthe above hexose oxidase preparation. The measurement was carried outessentially in accordance with the colorimetric method of Ellman (1958)as also described in Cereal Chemistry, 1983, 70, 22-26. This method isbased on the principle that 5.5′-dithio-bis(2-nitrobenzoic acid) (DTNB)reacts with thiol groups in the dough to form a highly coloured anion of2-nitro-5-mercapto-benzoic acid, which is measuredspectrophotometrically at 412 nm.

[0208] Assuming that the relative change of the amount of thiol groupsin a dough is reflected as the change in the optical density (OD)resulting from the reaction between thiol groups and DTNB in the dough,the following results were obtained: Hexose oxidase GOD units/kg flourOD₄₁₂ 0 0.297 100 0.285 250 0.265 875 0.187 1250 0.138

[0209] Thus, this experiment showed a significant decrease in ODindicating a reduction of the content of free thiol groups which wasproportionate to the amount of hexose oxidase activity added.

[0210] 2.4. Improvement of the Rheological Characteristics of Dough bythe Addition of Hexose Oxidase

[0211] The above dough was subjected to extensigraph measurementsaccording to AACC Method 54-10 with and without the addition of anamount of the hexose oxidase preparation corresponding to 100 units/kgflour of hexose oxidase activity. The dough without addition of enzymeserved as a control.

[0212] The principle of the above method is that the dough after formingis subjected to a load-extension test after resting at 30° C. for 45,90, 135 and 180 minutes, respectively, using an extensigraph capable ofrecording a load-extension curve (extensigram) which is an indication ofthe doughs resistance to physical deformation when stretched. From thiscurve, the resistance to extension, B (height of curve) and theextensibility, C (total length of curve) can be calculated. The B/Cratio (D) is an indication of the baking strength of the flour dough.

[0213] The results of the experiment is summarized in Table 2.1 below.TABLE 2.1 Extensigraph measurements of dough supplemented with 100 GODunits/kq flour of hexose oxidase (HOX). Sample Time, min B C D = B/CControl 45 230 180 1.3 HOX 45 320 180 1.8 Control 90 290 161 1.8 HOX 90450 148 3.0 Control 135 290 167 1.7 HOX 135 490 146 3.4 Control 180 300168 1.8 HOX 180 500 154 3.2

[0214] It is apparent from this table that the addition of hexoseoxidase (HOX) has an improving effect on the doughs resistance toextension as indicated by the increase in B-values. This is reflected inalmost a doubling of the B/C ratio as a clear indication that the bakingstrength of the flour is significantly enhanced by the hexose oxidaseaddition.

[0215] In a similar experiment, 100 units/kg flour of a commercialglucose oxidase product was added and the above parameters measured inthe same manner using a dough without enzyme addition as a control. Theresults of this experiment is shown in Table 2.2 below: TABLE 2.2Extensigraph measurements of dough supplemented with 100 GOD units/kgflour of glucose oxidase (GOX). Sample Time, min B C D = B/C Control 45240 180 1.3 GOX 45 290 170 1.7 Control 90 260 175 1.5 GOX 90 360 156 2.3Control 135 270 171 1.6 GOX 135 420 141 3.0

[0216] When the results for the above two experiments are compared withregard to differences between control dough and the hexose oxidase orglucose oxidase supplemented doughs it appeared that hexose oxidase hasa stronger strengthening effect than glucose oxidase. Furthermore, theB/C ratio increased more rapidly with hexose oxidase relative to glucoseoxidase which is a clear indication that enhancement of the bakingstrength is being conferred more efficiently by hexose oxidase than byglucose oxidase.

EXAMPLE 3

[0217] Dough Improving Effect of Hexose Oxidase Extracted from Chondruscrispus

[0218] For this experiment fresh Chondrus crispus seaweed fronds wereharvested along the coast of Hirsholmene, Denmark. Hexose oxidase wasisolated using two different extraction procedures, and the materialsfrom both were pooled for the below dough improving experiment.

[0219] 3.1. Purification of Hexose Oxidase from Chondrus crispus I

[0220] 954 g of the fresh fronds was rinsed in distilled water, driedwith a towel and stored in liquid nitrogen. The seaweed was blendedusing a Waring blender and 1908 ml of 0.1 M sodium phosphate buffer, 1 MNaCl, pH 6.8 was added to the blended seaweed. The mixture was extractedunder constant stirring for 4 days at 5° C., followed by centrifugationof the mixture at 20,000×g for 30 minutes.

[0221] The resulting 1910 ml supernatant (351.1 U/ml) was concentratedto 440 ml at 40° C. in a Buchi Rotavapor R110. The concentrate wasammonium sulphate fractionated to 25% The mixture was stirred for 30minutes and centrifuged for 20 minutes at 47,000×g. The supernatant (395ml) was dialysed overnight against 20 l of 10 mM triethanolamine (TEA)buffer, pH 7.3 to a final volume of 610 ml (367.1 U/ml).

[0222] The above 610 ml was applied in two runs to a 2.6×25 cm columnwith 130 ml Q-Sepharose FF equilibrated in 20 mM TEA buffer, pH 7.3. Thecolumn was washed with the equilibration buffer and the bound proteinswere eluted using 800 ml gra dient from 0 to 0.8 M NaCl in equilibrationbuffer. The column was eluted at 4 ml/minute and fractions of 12 mlcollected. Fractions containing the hexose oxidase activity werecollected and pooled to a final volume of 545 ml (241.4 U/ml).

[0223] 3.2. Purification of Hexose Oxidase from Chondrus crispus II

[0224] 1250 g of the fresh fronds was rinsed in distilled water, driedwith a towel and stored in liquid nitrogen. The seaweed was blended in aWaring blender followed by the addition of 2500 ml 0.1 M sodiumphosphate buffer, 1 M NaCl. pH 6.8. The mixture was extracted undercontinuous stirring for 4 days at 5° C. followed by centrifugation at20,000×g for 30 minutes.

[0225] The resulting 2200 ml supernatant (332.8 U/ml) was concentratedto 445 ml at 40° C. using a Buchi Rotavapor R110. The resultingconcentrate was ammonium sulphate fractionated to 25%. The mixture wasstirred for 30 minutes and centrifuged for 20 minutes at 47,000×g. Theprecipitate was discarded. The 380 ml supernatant was dialysed overnightagainst 20 l 10 mM TEA buffer, pH 7.3, to a final volume of 850 ml(319.2 U/ml).

[0226] The above 850 ml was applied to a 2.6×25 cm column with 130 mlQ-Sepharose FF equilibrated in 20 mM TEA buffer, pH 7.3.

[0227] The column was washed with the equilibration buffer and the boundproteins were eluted using 800 ml gradient from 0 to 0.8 M NaCl inequilibration buffer. The column was eluted at 4 ml/minute and fractionsof 12 ml collected. Fractions containing the hexose oxidase activitywere collected and pooled to a final volume of 288 ml. The retentatefrom the above step was applied to a 2.6×31 cm column with 185 ml metalchelating sepharose FF loaded with Ni²⁺ and equilibrated in 50 mM sodiumphosphate, 1 M NaCl, pH 7.4. The bound proteins were eluted with a 740ml gradient of 0 to 35 mM imidazole, pH 4.7 in equilibration buffer. Thecolumn was eluted at 2 ml/minute and fractions of 11 ml was collected.Fractions 41-54 (140 ml, 352.3 U/ml) were pooled. Some hexose oxidasedid run through the column.

[0228] 3.3. Pooling and Concentrating of Extracts

[0229] The run through and the 140 ml from purification II and the 545ml from purification I were pooled to a final volume of 1120 ml (303.6U/ml). The 1120 ml was rotation evaporated into a volume of 210 mlfollowed by dialysis overnight 30 against 20 l of 10 mM TEA buffer, pH7.3, to a final volume of 207 ml (1200.4 U/ml)

[0230] 3.3.1. Anion Exchange Step

[0231] The retentate resulting from the above step was applied to a2.6×25 cm column with 130 ml Q-sepharose FF equilibrated in 20 mMtriethanolamine, pH 7.3. The column was washed with the equilibrationbuffer and the bound proteins eluted using 800 ml gradient from 0 to 0.8M NaCl in equilibration buffer. The column was eluted at 4 ml/minute andfractions of 12 ml collected. Fractions 30-50 containing the hexoseoxidase activity (260 ml, 764.1 U/ml) were collected and pooled.

[0232] 3.3.2. Other Enzyme Activity

[0233] The above pooled solution was tested for the following enzymaticside activities catalase, protease, xylanase, α- and β-amylase andlipase. None of these activities were found in the solution.

[0234] 3.4. Improvement of the Rheological Characteristics of Dough bythe Addition of Hexose Oxidase

[0235] A dough was prepared from wheat flour, water and salt and 0, 72,216 and 360 units per kg of flour, respectively of the above hexoseoxidase preparation was added hereto. The dough without addition ofenzyme served as a control. In addition two doughs were prepared towhich was added 216 and 360 units per kg of flour respectively, ofGluzyme, a glucose oxidase available from Novo Nordisk A/S. Denmark.

[0236] The doughs were subjected to extensigraph measurements accordingto a modification of the above AACC Method 54-10. The results of theexperiment are summarized in Table 3.1 below. TABLE 3.1 Extensigraphmeasurements of dough supplemented with hexose oxidase (HOX) or glucoseoxidase (units per kg flour) Time, Sample Min B C D = B/C Control 45 250158 1.6 HOX 72 U/kg 45 330 156 2.1 HOX 216 U/kg 45 460 153 3.0 HOX 360U/kg 45 580 130 4.5 Gluzyme 72 U/kg 45 350 159 2.2 Gluzyme 216 U/kg 45340 148 2.3 Gluzyme 360 U/kg 45 480 157 3.1 Control 90 290 164 1.8 HOX72 U/kg 90 470 145 3.2 HOX 216 U/kg 90 650 142 4.6 HOX 360 U/kg 90 870116 7.5 Gluzyme 72 U/kg 90 450 147 3.1 Gluzyme 216 U/kg 90 480 138 3.5Gluzyme 360 U/kg 90 500 152 3.2 Control 135 330 156 2.1 HOX 72 U/kg 135540 129 4.2 HOX 216 U/kg 135 750 125 6.0 HOX 360 U/kg 135 880 117 7.5Gluzyme 72 U/kg 135 510 136 3.8 Gluzyme 216 U/kg 135 550 122 4.5 Gluzyme360 U/kg 135 560 121 4.6

[0237] It is evident from the above table that the addition of hexoseoxidase (HOX) or glucose oxidase had an improving effect on theresistance of doughs to extension as indicated by the increase inB-values. This is reflected in an increase of the B/C ratio as a clearindication that the baking strength of the flour was enhancedsignificantly by the addition of enzymes.

[0238] It is also evident that the hexose oxidase had a higherstrengthening effect than glucose oxidase. Furthermore, the B/C ratioincreased more rapidly with hexose oxidase relative to glucose oxidasewhich is a clear indication that enhancement of the baking strength isbeing conferred more efficiently by hexose oxidase than by glucoseoxidase.

EXAMPLE 4

[0239] Dough Improving Effect of Hexose Oxidase Extracted from Chondruscrispus

[0240] 4.1. Purification of Hexose Oxidase from Chondrus crispus

[0241] Fresh Chondrus crispus fronds were harvested along the coast ofBrittany, France. 2285 g of this fresh material was rinsed in distilledwater, dried with a towel and stored in liquid nitrogen. The seaweed wasblended in a Waring blender followed by addition of 4570 ml 0.1 M sodiumphosphate buffer, 1 M NaCl pH 6.8. The mixture was extracted undercontinuous magnetic stirring for 4 days at 5° C. followed bycentrifugation at 20,000×g for 30 minutes.

[0242] The resulting 4930 ml supernatant (624.4 U/ml) was concentratedto 1508 ml at 40° C. using a Buchi Rotavapor Rll0. The obtainedconcentrate was polyethylenglycol fractionated to 3% (w/v). The mixturewas stirred for 30 minutes and centrifuged for 30 minutes at 47,000×g.The pellet was discarded. The 1470 ml supernatant (2118.7 U/ml) was PEGfractionated to 24%. The mixture was stirred for 30 minutes andcentrifuged for 30 minutes at 47,000×g. The supernatant was discardedand the 414.15 g of precipitate was resuspended in 200 ml 20 mM TEAbuffer, pH 7.3, followed by dialysis over night at 5° C. against 20 l 10mM TEA buffer, pH 7.3.

[0243] After dialysis the volume was 650 ml (2968.6 U/ml). Thesuspension was centrifuged for 30 minutes at 20,000×g. The precipitatewas discarded and the supernatant was diluted to 3200 ml with distilledwater.

[0244] The above 3200 ml (829.9 U/ml) was applied to a 10×14 cm columnwith 1100 ml Q-Sepharose FF equilibrated in 20 mM TEA buffer, pH 7.3.The column was washed with the equilibration buffer and the boundproteins were eluted using 15,000 ml gradient from 0 to 0.8 M NaCl inequilibration buffer. The column was eluted at 50 ml/minute. Hexoseoxidase did run through the column and 840 ml of this was collected.

[0245] The 840 ml suspension was treated with kieselguhr andconcentrated to 335 ml (2693.3 U/ml).

[0246] The above 335 ml was applied to a 3 l Sephadex G25C desaltingcolumn 10×40 cm. The column was equilibrated in 20 mM TEA buffer, pH7.3, eluted at a flow rate of 100 ml/minute and 970 ml eluate wascollected. This eluate was applied to a 10×14 cm column with 1100 mlQ-Sepharose FF equilibrated in 20 mM TEA, pH 7.3. The column was washedwith the equilibration buffer and bound proteins eluted using a 15,000ml gradient of 0 to 0.8 M NaCl in equilibration buffer. The column waseluted at 50 ml/min. Hexose oxidase did run through the column and 1035ml of this was collected.

[0247] To the above eluate (1035 ml) ammonium sulphate was added to afinal concentration of 2 M. The mixture was then applied in two runs toa 5×10 cm column with 200 ml phenyl sepharose HP equilibrated in 25 mMsodium phosphate buffer, pH 6.3 and 2 M (NH₄)₂SO₄. The column was washedwith equilibration buffer followed by eluting the bound proteins at aflow rate of 50 ml/minute using 5,000 ml gradient from 2 M to 0 M(NH₄)₂SO₄ in 25 mM sodium phosphate buffer. Fractions of 500 and 29 ml,respectively were collected from run 1 and 2. Fraction 5 in run 1 andfractions 27-42 in run 2 containing the hexose oxidase activity werepooled to a total of 1050 ml (563.9 U/ml).

[0248] The above pool was desalted by a 3 1 Sephadex G25C gel filtrationcolumn. The column was equilibrated in 20 mM TEA buffer, pH 7.3, elutedat a flow rate of 100 ml/minute and 1,000 ml eluate was collected.

[0249] The 1,000 ml eluate was concentrated to 202 ml (2310.2 U/ml) andthis preparation was used for following rheology testing.

[0250] 4.2. Improvement of the Rheological Characteristics of Dough bythe Addition of Hexose Oxidase

[0251] A dough was prepared from wheat flour, water and salt and 0, 288,504 and 720 oxidoreductase units per kg of flour, respectively of theabove hexose oxidase preparation was added hereto. The dough withoutaddition of enzyme served as a control. In addition two doughs wereprepared to which was added 288 and 504 oxidoreductase units per kg offlour respectively, of Gluzyme, a glucose oxidase available from NovoNordisk A/S. Denmark.

[0252] The doughs were subjected to extensigraph measurements accordingto a modification of AACC Method 54-10.

[0253] The results of the experiment are summarized in Table 4.1 below.TABLE 4.1 Extensigraph measurements of dough supplemented with hexoseoxidase (HOX) or glucose oxidase (Units per kg flour) Time, Sample Min BC D = B/C Control 45 210 171 1.2 HOX 288 U/kg 45 490 139 3.5 HOX 504U/kg 45 640 122 5.2 HOX 720 U/kg 45 730 109 6.7 Gluzyme 288 U/kg 45 350165 2.1 Gluzyme 504 U/kg 45 385 153 2.5 Gluzyme 720 U/kg 45 435 148 2.9Control 90 275 182 1.5 HOX 288 U/kg 90 710 130 5.5 HOX 504 U/kg 90 825106 7.8 HOX 720 U/kg 90 905 107 8.5 Gluzyme 288 U/kg 90 465 153 3.0Gluzyme 504 U/kg 90 515 135 3.8 Gluzyme 720 U/kg 90 540 140 3.9 Control135 280 175 1.6 HOX 288 U/kg 135 745 102 7.3 HOX 504 U/kg 135 920 94 9.8HOX 720 U/kg 135 — 80 — Gluzyme 288 U/kg 135 525 129 4.1 Gluzyme 504U/kg 135 595 129 4.6 Gluzyme 720 U/kg 135 630 121 5.2

[0254] It is apparent from the above results that the addition of hexoseoxidase (HOX) or glucose oxidase has an improving effect on theresistance of doughs to extension as indicated by the increase inB-values. This is reflected in an increase of the B/C ratio.

[0255] It is also apparent that hexose oxidase has a stronger sstrengthening effect than that of glucose oxidase, the strengtheningeffect of both enzymes being proportional to the amount of enzyme added.Furthermore, the B/C ratio increased more rapidly with hexose oxidaserelative to glucose oxidase which is a clear indication that enhancementof the baking strength is being conferred more efficiently by hexoseoxidase than by glucose oxidase.

EXAMPLE 5

[0256] Improving Effect of Hexose Oxidase Extracted from Chondruscrispus on the Specific Volume of Bread

[0257] 5.1. Purification of Hexose Oxidase from Chondrus crispus

[0258] Fresh Chondrus crispus fronds were harvested along the coast ofBrittany, France. 2191 g of this fresh material was rinsed in distilledwater, dried with a towel and stored in liquid nitrogen. The seaweed wasblended in a Waring blender fol lowed by addition of 4382 ml 0.1 Msodium phosphate buffer, 1 M NaCl and pH 6.8. The mixture was extractedunder continuously magnetic stirring for 4 days at 5° C. followed bycentrifugation at 20,000×g for 20 minutes.

[0259] The resulting 4600 ml supernatant (746.1 U/ml) was concentratedto 850 ml at 40° C. in a Buchi Rotavapor R110. This concentrate (3626.9U/ml) was polyethylene glycol fractionated to 3% (w/v). The mixture wasstirred for 30 minutes and centrifuged for 30 minutes at 20,000×g. Theprecipitate was discarded. The 705 ml supernatant (2489.8 U/ml) was PEGfractionated to 25%. The mixture was stirred for 30 minutes andcentrifuged for 30 minutes at 20,000×g. The supernatant was discardedand the 341 g of precipitate was resuspended in 225 ml 20 mM TEA buffer,pH 7.3. The suspension (500 ml) was desalted on a 3 l Sephadex G25Cdesalting column 10×40 cm. The column was equilibrated in 20 mM TEAbuffer, pH 7.3, and eluted at a flow rate of 100 ml/minute. 1605 mleluate was collected.

[0260] To the above eluate (687.5 U/ml) ammonium sulphate was added to afinal concentration of 2M. The mixture was then applied in two runs to a5×10 cm column with 200 ml phenyl sepharose HP equilibrated in 25 mMsodium phosphate buffer, pH 6.3 and 2 M (NH₄)₂SO₄. The column was washedwith equilibration buffer followed by elution of the bound proteins at aflow rate of 50 ml/minute using 5,000 ml gradient from 2 M to 0 M(NH₄)₂SO₄ in 25 mM sodium phosphate buffer. Fractions of 29 ml wascollected. Fractions 85-105 in run 1 and fractions 36-69 in run 2containing the hexose activity were pooled to a total of 1485 ml (194.7U/ml).

[0261] The above pool was desalted by a 3 l Sephadex G25C gelfiltrationcolumn, the same as used in 4.1. The column was equilibrated in 20 mMTEA buffer, pH 7.3, and eluted at a flow rate of 100 ml/minute. 1,200 mleluate was collected.

[0262] The 1,200 ml eluate was concentrated to 685 ml (726.2 U/ml) andused for baking experiments.

[0263] 5.2. Improvement of the Specific Volume of Bread by Adding HexoseOxidase to the Dough

[0264] A dough was prepared from 1500 g of flour, 90 g of yeast, 24 g ofsalt, 24 g of sugar and 400 BU of water and 0 or 108 units of the abovepurified hexose oxidase and 108 units of Gluzyme (glucose oxidaseavailable from Novo Nordisk, Denmark) per kg flour, respectively wasadded hereto. The dough was mixed on a Hobart mixer for 2+9 minutes at26° C. and divided into two parts followed by resting for 10 minutes at30° C. in a heating cabinet, moulding with a Fortuna 3/17/7 and proofingfor 45 minutes at 34° C. and 85% RH. The thus proofed dough was baked at220° C. for 17 minutes with 12 sec. steam in a Bago oven.

[0265] The results of the experiment are summarized in table 5.1 below.TABLE 5.1 Improvement of specific volumes of bread prepared from doughsupplemented with hexose oxidase or glucose oxidase (Units per kg flour)Total Total Specific volume weight volume control 5325 1027 5.18 Hexoseoxidase 108 U/kg 6650 1036 6.41 Gluzyme 108 U/kg 6075 1030 5.89

[0266] It is evident from the above table that the addition of hexoseoxidase or glucose oxidase had an increasing effect on the total volume,the weight being essentially the same. This is reflected in an increaseof the specific volume as compared to the bread baked without additionof enzymes.

[0267] It is also evident that hexose oxidase has a significantly largereffect on the increase of the specific volume than had glucose oxidaseat the same dosage.

EXAMPLE 6

[0268] Characterization of the Purified Hexose Oxidase

[0269] Preparations from the above purifications were used forcharacterization of hexose oxidase.

[0270] 6.1. Staining for Hexose Activity After Non-Denaturing PAGE

[0271] Hexose oxidase activity was analyzed by native PAGE using precast8-16% Tris-glycine Novex gels according to the manufactures instructions(Novex, San Diego, USA). After electrophoresis the gels were stained forhexose oxidase activity by incubation of the gel in a solutioncontaining 50 mM sodium phosphate buffer, pH 6.0, 100 mM glucose, 50mg/l phenazine methosulphate (Sigma P9625) and 250 mg/l nitrobluetetrazolium (Sigma N6876) as described in the PhD thesis by Witteveen,C. F. B. (1993) “Gluconate formation and polyol metabolism inAspergillus niger”. After about 30 minutes the hexose oxidase activitywas visible as a double band very close to each other. The same doubleband was also seen when a native PAGE of hexose oxidase was silverstained. The molecular weight of purified hexose oxidase was determinedto 144 kD by native PAGE. Half the gel was silver stained, the otherhalf was activity stained. As standards were used bovine serum albumin(67 kD), lactate dehydrogenase (140 kD), catalase (232 kD), ferritin(440 kD) and thyroglobulin (669 kD).

[0272] 6.2 Determination of Molecular Weight by SDS-Page

[0273] The molecular weight was also determined on material which wasfirst applied to a native PAGE as described above, after activitystaining the hexose oxidase band was excised from the gel and thenelectroeluted using an Electro-Eluter (model 422, Bio-Rad, CA, USA)according to the manufacturer's recommendations. The electroelutedprotein was subjected to SDSPAGE and silver stained. This material gave“one” double band at about 70 kDa in SDS-PAGE gels. The electroelutedhexose oxidase is therefore a dimer of two subunits.

[0274] 6.3 Determination of pI of Hexose Oxidase

[0275] Samples containing hexose oxidase activity were analyzed byisoelectric focusing (IEF) using a precast 3-10 IEF gel according to themanufacturer's recommendations (Novex, San Diego, US). Afterelectrophoresis half of the gel was silver stained and the other halfnitroblue tetrazolium stained as described in 6.1.

[0276] Hexose oxidase stained as a double band. The pI of the first bandwas 4.79, pI of the second band was 4.64. As standards were usedtrypsinogen (9.30), lentil lectin basic band (8.65), lentil lectinmiddle band (8.45), lentil lectin acid band (8.15), horse myoglobinacidic band (6.85), human carbonic anhydrase B (5.85), β-lactoglobulin A(5.20), soy bean trypsin inhibitor (4.55) and amyloglucosidase (3.50).

[0277] 6.4 Determination of Km Hexose Oxidase for Different Sugars

[0278] Km of hexose oxidase was determined for 7 different sugars asdescribed in 1.2.3. Results are summarized in table 6.1 below. TABLE 6.1Determination of Km of hexose oxidase for different sugars Substrate Km(mM) cv (mM) D-glucose 2.7 0.7 D-galactose 3.6 1 cellobiose 20.2 7.8maltose 43.7 5.6 lactose 90.3 20.6 xylose 102 26 arabinose 531 158

[0279] 6.5 Determination of a Peptide Sequence of Hexose Oxidase

[0280] 50 μl from the electroeluted mixture in 6.2 was suspended in 450μl 0.1% triflouracetic acid (TFA).

[0281] To remove the Tris, glycine and SDS, the above mixture wassubjected to chromatography on reverse-phase HPLC. The resultingsolution was applied in 9 runs to a 4.6×30 cm Brownlee C2 columnequilibrated in 0.1% TFA. The column was washed in equilibration bufferand bound peptides eluted with a 14 ml gradient from 10 to 80%acetonitrile in 0.1% TFA, at a flow rate of 0.7 ml/min. Fractions fromthe largest peak containing the enzyme were collected and freeze dried.

[0282] 6.5.1 Endoproteinase Lys-C Digestion

[0283] The resulting freeze dried enzyme was dissolved in 50 μl 8 Murea, 0.4 M NH₄HCO₃, pH 8.4. Denaturation and reduction of the proteinwas carried out by the addition of 5 μl 45 mM di-thiothreitol and underan overlay of N₂ at 50° C. for 15 min. The solution was cooled to roomtemperature and 5 μl 100 mM iodoacetamide was added, the cysteines beingderivatized for 15 min. at room temperature in the dark under N₂.Subsequently, the solution was suspended in 135 μl water and digestionwas carried out at 37° C. under N₂ for 24 hours by addition of 5 μgendoproteinase Lys-C dissolved in 5 l water. The reaction was terminatedby freezing the reaction mixture at −20° C.

[0284] 6.5.2 Reverse-Phase HPLC Separation of Peptides

[0285] The resulting peptides were separated by reverse-phase HPLC on aVYDAC c18 column 0.46×15 cm (The Separation Group, CA, USA) using assolvent A 0.1% TFA in water and as solvent B 0.1% TFA in acetonitrile.

[0286] 6.5.3 Peptide Sequencing

[0287] Sequencing was performed on an Applied Biosystems 476A sequencer(Applied Biosystems, CA, USA) using pulsed-liquid fast cycles accordingto the manufacturer's instructions. A peptide having the below aminoacid sequence was identified:

[0288] D P C Y I V I D V N A G T P O K P D P.

EXAMPLES 7 TO 10

[0289] Definitions

[0290] All PANODAN™ products contain DATEM (Di-acetyl tartaric acidester of monoglycerides) and are obtained from Danisco A/S.

[0291] PANODAN™ 521: DATEM containing bacterial xylanase and fungalamylase

[0292] TS-E 662™ (obtained from Danisco A/S) is a product containinghexose oxidase (Hox) (EC 1.1.1.5) from Chondrus chrispus expressed inHansenula polymorpha.

[0293] TS-E 680™ (obtained from Danisco A/S) is a product containingfungal xylanase (EC 3.2.1.8) from Aspergillus niger.

[0294] TS-E 861™ (obtained from Danisco A/S) is a product containingfungal xylanase (EC 3.2.1.8) from Aspergillus niger, lipase (EC 3.1.1.3)from Thermomyces lanuginosa expressed in Aspergillus oryzae, and hexoseoxidase (EC 1.1.1.5) from Chondrus crispus expressed in Hansenulapolymorepha.

[0295] GRINDAMYL™ H 640 (obtained from Danisco A/S): contains bacterialxylanase

[0296] Grindamyl™ H 121 (obtained from Danisco A/S) is a fungal xylanase(EC 3.2.1.8) from Aspergillus niger.

[0297] Grindamyl™ EXEL 16 (obtained from Danisco A/S) is lipase (EC3.1.1.3) from Thermomyces lanuginosa expressed in Aspergillus oryzae.

[0298] Grindamyl™ EXEL 66 (obtained from Danisco A/S) is a mixture oflipase (EC 3.1.1.3) from Thermomyces lanuginosa expressed in Aspergillusoryzae and a fungal xylanase (EC 3.2.1.8) from Aspergillus niger.

[0299] Lipopan F™ (Lipopan F BG) (obtained from Novozymes) is accordingto its producer (Novozymes) a purified lipolytic enzyme from Fusariumoxysporum produced by submerged fermentation of a genetically modifiedAspergillus oryzae microorganism. According to its producer, Lipopan Fhas inherent activity toward phospholipids, glycolipids andtriglycerides.

[0300] Recipes/Procedures

[0301] High Volume Tweedy

[0302] Recipe Product Name % Gram ppm Ijsvogel flour 3000 Water 58 Salt60 Compressed yeast 180 Ascorbic acid 30

[0303] Procedure:

[0304] Dough temperature: 29° C. (dough temp.−flour temp.+4° C. watertemp.)

[0305] Mixing: 55 WH no vacuum

[0306] Resting: 5 min. at room temperature

[0307] Scaling: 500 g (bread), 1350 g (rolls)

[0308] Resting: 5 min. at room temperature

[0309] Moulding: Puma I 13 II 18 (bread), Fortuna 3/17/7 (rolls), Glimek(moulding machine) 1:4, 2:3, 3:12, 4:14

[0310] Proofing: 70 min. at 43° C., 70% RH. (bread), 50 min. at 34° C.,85% RH. (rolls)

[0311] Baking: BAGO, 35 min.+5 min. with the steamer open at 220° C., 12sec. steam (bread), 17 min. at 220° C., 17 sec. steam (rolls)

[0312] Turkish Batard

[0313] Recipe Product name % Gram ppm Ijsvogel flour 2000 Water 57,00Compressed yeast 80 Salt 30 Ascorbic acid 70

[0314] Procedure:

[0315] Flour temperature: 15-17° C. (for trials—storage day before useat 15° C.)

[0316] Mixing: 35 min. After 25 min. add salt

[0317] After 30 min. add yeast

[0318] Dough temp.: 23-25° C.

[0319] Resting: 30 min. Bulk rest on table (table=22° C. & 80% RH)

[0320] Scaling 300 g. pieces

[0321] Rounding: By hand

[0322] Resting: 25 min. on table (table=22° C. & 80% RH) . . . startclock when scaling starts

[0323] Molding=Glimek: 1:5, 2:4, 3:15, 4:10 . . . 10 in innerpos.

[0324] Proofing: 60 min. & 90 min. for this trial at 30° C. & 85% RH

[0325] Shock test

[0326] Baking: 20 min. in Bago1 & 25 min. in Bago2 . . . the last 5 min.is with the damper open for both ovens.

[0327] Bago1: 250° C. start temp. 5 sec. steam with damper open. Oventemp. down to 230° C. at once. Close damper after 11% min.

[0328] Bago2: 275° C. start temp. 8 sec. steam with damper open. Oventemp. down to 260° C. at once. Close damper after 11% min.

[0329] Crispy Rolls

[0330] Recipe: Product Name % Gram ppm Danish silver flour 2000 Water58/60 Compressed yeast 120 Salt 32 Sugar 32 Ascorbic acid 40

[0331] Procedure:

[0332] Mixing: Diosna 2+5 min. (depending on flour)

[0333] Dough temperature: 26° C.

[0334] Scaling: 1350 g

[0335] Resting: 10 min. at 30° C. in heating cabinet

[0336] Moulding: Fortuna 3/17/7

[0337] Proofing: 45 min alternatively 90 min at 34° C., 85% RH.

[0338] Baking: 18 min. at 220° C., 8 sec. steam (Bago-oven), 7 sec.steam (Wachtel-oven)

[0339] (MIWE program 28) (0.35 litre steam, 15 min. at 2000° C., ½ min.at 2200° C.)

[0340] US Toast

[0341] Here a sponge as a pre-mix is prepared, to all of which is thenadded the dough. Recipe Gr % US Flour 900.000 g 50.000% Sponge: Water900.000 g 50.000% Dry  23.400 g 1.300% Yeast Yeast Food  5.400 g 0.300%Enzyme  0.054 g 0.003% Complex ADA  0.036 g 0.002% US Flour 900.000 g50.000% Dough: Water 234.000 g 13.000% Dry  25.200 g 1.400% Yeast Sugar153.000 g 8.500% Salt  43.200 g 2.400% Shortening (fat)  36.000 g 2.000%Sod.Prop.  8.100 g 0.450% Dimodan SDM-T (P100/B)  9.000 g 0.500% Asc.Acid.  0.072 g 0.004% ′→ (=7,200 g to 1000 ml. Take 10 ml. from thesolution)

[0342] Datem 22-CA-60 4.500 g 0.2500% S685 300 PPM H640 20 PPM TS-E 662100 PPM

[0343] Care has to be taken with the water amount added from asc. acidsolution and other water based solutions ex. enzymes.

[0344] The extra added water amount should be be withdrawn from thewater amount on the Dough-side of the recipe.

[0345] The enzyme complex is a mix of alpha amylase andamyloclucosidase.

[0346] DIMODAN SDM-T (P100/B) (obtained from Danisco A/S) is a distilledmonoglyceride.

[0347] Procedure: For the Sponge: Water Temp.: 25° C. Hobart mixer Step1, 1 min. Step 2, 1 min. Step 3, 1 min.

[0348] Fermentation: 2 h & 15 min. 40° C. & 80% RH (relative humidity)45 min. in freezer.

[0349] For the Dough:

[0350] Mix all ingredients together

[0351] Diosna-Mixer: Speed 1, 120 secs & Speed 2, 450 secs (or 28degrees dough temp.)

[0352] On table—rest 5 min.

[0353] Weigh out the breads at 450 g pr. bread—rest 5 min.

[0354] Glimek (moulding machine) adjustments: 1, 2, 14, 11—& 9 cm—readon outer position.

[0355] Fermentation:

[0356] 1 h & 10 min. 45 degrees Celsius & 90% RH

[0357] Bake-off:

[0358] Start temp.=250 degrees Celsius in 25 min.

[0359] Insert the breads and adjust bake-off temperature to 200 degreesCelsius at once.

[0360] Baking Trials

[0361] In each trial the dough characteristic, stickiness and all overbread score have been evaluated. The dough characteristic is a total ofthree different parameters: dough extensibility evaluated just aftermixing and again after resting and stickiness after resting. Eachparameter has been evaluated by bakers on a scale from 1-10, where 10are the best. The score in the examples are a total of these differentscores.

[0362] Stickiness evaluation has been subjectively evaluated by bakersjust after mixing on a scale from 1 to 10, where 10 is the best, meaningnon sticky.

[0363] All over bread score is a total of an evaluation made on breadcrust, -crumb, possible capping and all over energy of the bread. Againeach parameter is evaluated on a scale from 1-10, where 10 is the best.

EXAMPLE 7 Testing Alternatives in Tweedy Bread (UK Procedure)

[0364] The breads were rested for 70 min each and after a full proofing,each bread was shock treated in order to evaluate the shock resistanceand thereby the dough stability.

[0365] In the baking trials, both pure enzyme solutions and combinationsof DATEM and enzymes were tested as alternative to Lipopan F.

[0366] Baking Trials 4969-29 Specific Shocked Dough Dough All over Testvolume, ccm/g volume, ccm/g characteristic stickiness bread score 0.4%PANODAN GB 5.6 4.64 15 4 29 0.2% PANODAN GB, 5.75 4.92 14 4 30 100 ppmGRINDAMYL H121, 100 ppm TS-E 662 100 ppm TS-E 662, 5.57 4.47 14 4 20 100ppm GRINDAMYL H121, 100 ppm GRINDAMYL EXEL 16 40 ppm Lipopan F 5.7 4.613 4 29 0.2% PANODAN GB, 5.88 4.6 14 4 27 20 ppm Lipopan F 20 ppmLipopan F, 5.65 4.78 14 4 29 100 ppm TS-E 662, 100 ppm GRINDAMYL H121 40ppm Lipopan F, 5.79 4.82 13 4 29 100 ppm TS-E 662, 100 ppm GRINDAMYLH121

[0367] From the results it can be concluded that PANODAN GB results in abetter crust of the product and a product.

[0368] The combination of PANODAN GB in combination with xylanase andhexose oxidase yields a beneficial effect.

[0369] When using DATEM and/or HOX in combination with GRINDAMYL EXEL 66the volume is increased significantly and the crust is considerablyimproved. The test with 0.1% PANODAN GB 100 ppm GRINDAMYL EXEL 66 and100 ppm TS-E 662 (HOX), gave a significantly good result at the samelevel as 0.4% PANODAN GB. Use of DATEM clearly gives a significantlypositive effect on the crust as compared to pure enzyme solutions.

EXAMPLE 8 Testing Alternatives in Turkish Batard

[0370] Baking Trials 7258-2 Specific Dough Dough All over volume,characteristic stickiness bread Test ccm/g * ** score*** 15 ppm LipopanF, 60 5.01 14 4 33 ppm TS-E 680 40 ppm Lipopan F 3.78 15 5 32 100 ppmTS-861* 5.03 16 5 44

[0371] Both from the specific volume in the table as well as thepictures shown in FIGS. 1-3 it can be concluded that TS-E 861 performsbetter.

EXAMPLE 9 Testing Alternatives in Crispy Rolls

[0372] The rolls were fermented at two different fermentation times—45and 90 min in order to stress the system and thereby give a betterpicture of the dough strengthening effect of the products. In general itcan be said that 90 min of fermentation for a small crispy roll is quitelong.

[0373] Baking Test: 4969-28 All Specific Specific Dough over volumevolume Dough sticki- bread 45 min, 90 min, characteristic ness scoreTest ccm/g ccm/g * ** *** 0.3% PANODAN 7.15 8.48 14 5 25 A2020 30 ppmLipopan F 6.83 8.1 14 4 26 100 ppm TS-E 662, 6.98 8.98 14 5 27 100 ppmGRINDAMYL H121, 100 ppm GRINDAMYL EXEL 16

[0374] From the results it can be seen that use of the combination ofxylanase, 1,3 triglyceride degrading lipase and hexose oxidase producesbeneficial results.

[0375] In short fermentation times (45 min.) at certain concentrationsPANODAN A2020 and Lipopan F gave comparable volume results. However,0.3% PANODAN A2020 showed better results with regard to crispiness ofthe crust and a better dough stability in general. We found that LipopanF often gave a slightly more “wet” crust.

[0376] Using HOX in combination with GRINDAMYL EXEL 66 and PANODAN 660results in an increase in dough stability.

[0377] With prolonged fermentation times (90 min.) all buns becomerelatively unstable. At some concentrations PANODAN A2020 does, however,give the best result.

EXAMPLE 10 Testing Alternatives in US Toast

[0378] Test of Lipopan F in a US sponge and dough using flour fromMexico—hard wheat type. The breads have all been fully proofed and afterthat each bread have been shock treated in order to evaluate the shockresistance and thereby the dough stability.

[0379] Baking Trials 7230-1: Specific Shocked Test volume, ccm/g volume,ccm/g 0.5% PANODAN 521 6.88 5.47 10 ppm Lipopan F 6.16 5.36 20 ppmLipopan F 6.44 5.30 40 ppm Lipopan F 6.28 5.52 0.25% PANODAN 521, 7.155.74 20 ppm GRINDAMYL H 640, 100 ppm TS-E 662

[0380] From these tests it is clear that the use of Hox results in a farbetter dough stability and consequently an increase of volume.

[0381] All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention may be apparent tothose skilled in the art without departing from the scope and spirit ofthe present invention. Although the present invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the claims.

[0382] Summary Paragraphs

[0383] Some aspects of the present invention are now described by way ofSummary Paragraphs.

[0384] 1. A method of improving the Theological properties of a flourdough and the quality of the finished product made from the dough,comprising adding to the dough ingredients, dough additives or the doughan effective amount of an oxidoreductase which is at least capable ofoxidizing maltose.

[0385] 2. A method according to paragraph 1 wherein the oxidoreductaseis hexose oxidase.

[0386] 3. A method according to paragraph 2 wherein the hexose oxidaseis derived from a source selected from an algal species, a plant speciesand a microbial species.

[0387] 4. A method according to paragraph 3 wherein the hexose oxidaseis derived from Chondrus crispus.

[0388] 5. A method according to paragraph 2 wherein hexose oxidase isadded in an amount which is in the range of 1 to 10,000 units per kg offlour.

[0389] 6. A method according to paragraph 5 wherein the hexose oxidaseis added in an amount which is in the range of 10 to 1000 units per kgof flour.

[0390] 7. A method according to paragraph 1 or 2 wherein the resistanceto extension of the dough in terms of the ratio between the resistanceto extension (height of curve, B) and the extensibility (length ofcurve, C), i.e. the B/C ratio, as measured by the AACC method 54-10 isincreased by at least 10% relative to that of an otherwise similar doughnot containing oxidoreductase.

[0391] 8. A method according to paragraph 1 wherein the finished productis bread.

[0392] 9. A method according to paragraph 1 wherein the finished productis a noodle product

[0393] 10. A method according to paragraph 1 wherein the finishedproduct is an alimentary paste product.

[0394] 11. A method according to paragraph 1 wherein at least onefurther enzyme is added to the dough ingredients, dough additives or thedough.

[0395] 12. A method according to paragraph 11 wherein the further enzymeis selected from the group consisting of a cellulase, a hemicellulase, axylanase, a starch degrading enzyme, a glucose oxidase, a lipase and aprotease.

[0396] 13. A dough improving composition comprising an oxidoreductasewhich is at least capable of oxidising maltose and at least one furtherdough ingredient or dough additive.

[0397] 14. A composition according to paragraph 13 wherein theoxidoreductase is derived from a source selected from an algal species,a plant species and a microbial species.

[0398] 15. A composition according to paragraph 14 wherein theoxidoreductase is hexose oxidase.

[0399] 16. A composition according to paragraph 15 wherein the hexoseoxidase is derived from Chondrus crispus.

[0400] 17. A composition according to paragraph 13 which is apre-mixture useful for preparing a baked product or in making a noodleproduct or an alimentary paste product.

[0401] 18. A composition according to paragraph 13 which comprises anadditive from the group consisting of an emulsifying agent and ahydrocolloid.

[0402] 19. A composition according to paragraph 18 wherein thehydrocolloid is selected from the group consisting of an alginate, acarrageenan, a pectin and a vegetable gum.

[0403] 20. A method of preparing a bakery product the method comprisingpreparing a flour dough to which is added an effective amount of anoxidoreductase which is at least capable of oxidising maltose, andbaking the dough.

[0404] 21. A method according to paragraph 20 wherein the specificvolume of the bakery product is increased relative to an otherwisesimilar bakery product prepared from a dough not containingoxidoreductase.

[0405] 22. A method according to paragraph 21 wherein the specificvolume is increased by at least 20%.

[0406] 23. A method according to paragraph 20 wherein at least onefurther enzyme is added to the dough.

[0407] 24. A method according to paragraph 20 wherein the further enzymeis selected from the group consisting of a cellulase, hemicellulase, axylanase, a starch degrading enzyme, a glucose oxidase, a lipase and aprotease.

[0408] 25. A method according to paragraph 20 wherein the oxidoreductaseis hexose oxidase.

[0409] 26. A method of preparing a flour dough-based food product,comprising adding to the dough an effective amount of a maltoseoxidising oxidoreductase.

[0410] 27. A method according to paragraph 26 wherein the oxidoreductaseis hexose oxidase.

What is claimed is:
 1. A method of improving the Theological and/ormachineability properties of a flour dough and/or the quality of theproduct made from the dough, comprising adding to the dough acombination comprising a Hox and an emulsifying agent.
 2. A methodaccording to claim 1 wherein the emulsifying agent is a lipase.
 3. Amethod according to claim 2 wherein the lipase comprises atriacylglycerol lipase, a galactolipase, or a phospholipase.
 4. A methodaccording to claim 1 wherein the Hox is isolated from a red algae.
 5. Amethod according to claim 1 wherein the flour dough comprises at leastone further dough additive or ingredient.
 6. A method according to claim5 wherein the further dough additive or ingredient is selected from thegroup consisting of a vegetable oil, a vegetable fat, an animal fat,shortening, butterfat, glycerol and milk fat.
 7. A method according toclaim 1 wherein the flour dough comprises a hard flour.
 8. A methodaccording to claim 1 wherein the product is a bread product.
 9. A methodaccording to claim 1 wherein at least one further enzyme is added to thedough ingredients, dough additives or the dough.
 10. A method accordingto claim 9 wherein the further enzyme comprises a xylanase, a cellulase,a hemicellulase, a starch degrading enzyme, a protease, a lipoxygenase,an oxidoreductase or a lipase.
 11. A dough improving compositioncomprising a Hox and an emulsifying agent.
 12. A dough improvingcomposition according to claim 11 wherein the emulsifying agent is alipase.
 13. A dough improving composition according to claim 12 whereinthe lipase comprises a triacylglycerol lipase, a galactolipase, or aphospholipase.
 14. A dough improving composition according to claim 13wherein the Hox is isolated from red algae.
 15. A dough improvingcomposition according to claims 11-14 wherein the flour dough comprisesat least one further dough additive or ingredient.
 16. A dough improvingcomposition according to claim 15 wherein the further dough additive oringredient comprises a vegetable oil, a vegetable fat, an animal fat,shortening, butterfat, glycerol or milk fat.
 17. A dough improvingcomposition according to claim 15 wherein the further dough additive oringredient is a hard wheat flour.
 18. A method of preparing a breadproduct comprising adding a dough improving composition according toclaims 11-14 to dough ingredients, dough additives or a dough and bakingthe dough comprising the dough improving composition to obtain the breadproduct.
 19. A dough improving composition according to claims 11-14wherein at least one further enzyme is added to the dough ingredients,dough additives or the dough.
 20. A dough improving compositionaccording to claim 19 wherein the further enzyme comprises a xylanase, acellulase, a hemicellulase, a starch degrading enzyme, a protease, alipoxygenase, an oxidoreductase or a lipase.
 21. A method of improvingthe Theological and/or machinability properties of a flour doughcomprising adding to the dough a dough improving composition of claim11.
 22. A method of improving the volume of a baked product made from aflour dough comprising adding to the dough a dough improving compositionof claim
 11. 23. A method of improving the Theological and/ormachineability properties of a flour dough and/or the quality of theproduct made from the dough, comprising adding to the dough acombination comprising a Hox and a triacylglycerol lipase.
 24. A methodof improving the rheological and/or machineability properties of a flourdough and/or the quality of the product made from the dough, comprisingadding to the dough a combination comprising a Hox and a galactolipase.25. A method of improving the Theological and/or machineabilityproperties of a flour dough and/or the quality of the product made fromthe dough, comprising adding to the dough a combination comprising a Hoxand a phospholipase.
 26. A method according to claim 9 wherein thefurther enzyme comprises a xylanase, an amylase or a mixture of axylanase and an amylase.
 27. A dough improving composition according toclaim 14 wherein the red algae comprises Iridophycus flaccidum, Chondruscrispus, or Euthora cristata.
 28. A dough improving composition of claim19, wherein the further enzyme comprises a xylanase, an amylase or amixture of a xylanase and an amylase.
 29. A method according to claim 4,wherein the red algae comprises Iridophycus flaccidum, Chondrus crispusor Euthora cristata.